Methods
Three hundred and fifty reverse transcriptase (RT) genotypes with at least three NRTI resistance mutations were included in this study; the corresponding strains were isolated from adolescents with a complex history of antiretroviral treatment. Subtyping was done using the publicly available algorithm REGA HIV-1&2. Resistance genotyping was performed using Big Dye Terminator chemistry provided by the ViroSeq genotyping system. The RT gene carrying the K65R mutation and thymidine analog mutations (TAMs) was cloned into pGEM-T vector (Promega), followed by sequencing. In order to identify mutational clusters we calculated the binomial (phi) correlation coefficient using SPSS 11.0 software.

Results
The analyzed sequences all belonged to the F1 subtype and were frequently carrying TAMs associated with substitutions at position 184. TAM-2 was the pathway more frequently encountered, and the demarcation between TAM-1 and TAM-2 was rather weak. Although the combination of K65R mutation with TAMs has rarely been reported because of their antagonistic effects on NRTI resistance, its presence was confirmed by clonal analysis of one strain. Four percent of the studied genotypes presented insertions and deletions in the region 67-70 of the RT gene and they were frequently associated with particular TAMs. Most of the NRTI resistance mutations were found to belong to one of three distinct clusters.

Conclusion
Although the overall resistance mutations were not different from those described for subtype B, the subtype F1 HIV-1 NRTI mutation patterns displayed same specificities with possible therapeutic consequences.